Nucleic acids, particularly single-stranded nucleic acids, can be separated by capillary electrophoresis. To perform a separation, a capillary tube is filled with a matrix. The matrix commonly comprises a polymer dissolved in a buffer solution and, sometimes, a denaturant. A sample volume is injected into one end of the capillary tube. Both ends of the capillary tube are immersed in a buffer solution and a large potential is applied across the capillary tube. The sample components are separated electrophoretically as they migrate through the capillary tube. Separation of the sample components within the matrix is based on the molecular size and charge of the injected species. Larger molecules migrate through the polymer matrix more slowly than smaller molecules. The polymer concentration in the matrix and/or degree of polymer crosslinking, if any, in the matrix can be varied to provide separation of species over a wide range of molecular weights and charges.
Polyacrylamide-based matrices provide high resolution for separating nucleic acid fragments, but are typically too viscous to rapidly load into or remove from a capillary tube. Furthermore, polyacrylamide matrices typically require capillary tubes that are coated on their inside walls to minimize or eliminate electroosmotic flow (EOF), which opposes the migration of the nucleic acid fragments. Excessively long migration times or in some cases loss of sample out the inlet side of the capillary can occur if the EOF is not reduced. The use of coated capillary tubes requires cumbersome preparation protocols and leads to reduced capillary life times due to coating degradation. Polyethylene oxide and hydroxyethyl cellulose are examples of other polymers that have been incorporated into matrices. Hydroxyethyl cellulose is preferably used with a capillary tube coated on its inside surface (See Bashkin et al., U.S. Pat. No. 5,534,123). (Hydroxypropyl)methyl cellulose (HPMC) based matrices have been used for double-stranded DNA separations with coated capillaries (See Cheng et al., Anal. Chem., 66 (1994) 4210). In addition, a HPMC-based matrix has been used for the capillary electrophoresis of double-stranded DNA in an uncoated capillary (See Shihabi, J. of Chromatography A, 853 (1999) 349–354). However, the separation matrices required for the efficient separation of double-stranded DNA differ significantly from those required for single-stranded DNA.
There is a need for a separation matrix that is easy to prepare, easy to load into a capillary tube, provides high resolution separation of single-stranded nucleic acid fragments, is easy to remove from the capillary tube and is transparent to ultraviolet radiation at 254 nm. Furthermore, the matrix should ideally work with fused silica capillary tubes with uncoated inside walls. Coated capillary tubes require extensive preparation and have shorter life times due to coating degradation.
This invention has as its primary objective to fulfull each of the above described needs. The method of accomplishing these as well as other objectives, both process and composition objectives, will be apparent from the detailed description.